De novo protein design has recently emerged as an attractive approach for studying the structure and function of proteins. This approach critically tests our understanding of the principles of protein folding; only in de novo design must one truly confront the issue of how to specify a protein's fold and function. If we truly understand proteins, it should be possible to design receptors, enzymes, and ion channels from scratch. Further, as this understanding evolves and is further refined, it should be possible to design proteins and biomimetic polymers with properties unprecedented in nature.

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

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Summary Many efforts are being devoted to the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization process and protein redesign. The considerable progress in these diverse design approaches have produced many metal-containing biocatalysts able to adopt functions of native enzymes or even novel functions beyond those found in Nature.

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.

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De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (Kdiss ‫؍‬ 0.001 fM), but the dissociation constant increases to 0.6 nM (⌬⌬G ‫؍‬ 5.4 kcal͞ mol monomer) as the active-site cavity is increased to accommodate small molecules.T he requirements for protein stability versus function are often diametrically opposed. The folded conformations of proteins are stabilized by maximizing the burial of hydrophobic groups, minimizing voids, and forming intramolecular hydrogen bonds (1, 2). In contrast, binding and enzymatic functions generally require active-site clefts replete with solventexposed hydrophobic groups and hydrogen-bonding groups, which are essential for proper binding of substrates. This tradeoff between conformational stability and function presents a particularly large challenge to protein design (3), forcing the designer to skirt the waters between Scylla and Charybdis.The stability͞function tradeoff is particularly apparent in metalloproteins. Structural metal-binding sites in proteins frequently achieve stability by binding metal ions in coordinately saturated ligand environments with idealized ligand-metal bond geometries (4, 5). However, functional sites in metalloenzymes frequently contain coordinately unsaturated metal ions that are positioned appropriately for binding substrates; they also sometimes display geometries not frequently observed in simple, small-molecule metal-ligand complexes (4, 5). Further, the active sites of metalloproteins are frequently preorganized in the absence of metal ions, which requires the burial of polar ligands at the expense of folding free energy (6). The preorganization imparts tight and geometrically specific metal binding by assuring that the protein imparts its own structural pr...